Abstract This research is to develop Fresnel-lens-based ultrasonic tweezers that can capture and manipulate living tissue in three dimensional (3D) space on demand, very much like optical tweezers but with several orders of magnitude stronger mechanical trapping force for a given temperature rise. The lab led by the Principal Investigator (PI) recently demonstrated ultrasonic capture of microparticles (70 - 400 m in diameter) in liquid with a single Multi-foci Fresnel Transducer (MFT). The captured microparticle was moved on demand by moving the MFT itself in 3D. Building on this success, this proposal will advance the transducer technology so that a single MFT can capture and move particles or cells in 3D on demand with an electrical signal without the need to move the transducer. This will benefit a wide range of biological researchers including those in molecular, developmental and cellular biology. Biological test experiments will be used to focus and validate the technology development. Our first specific application of the ultrasonic tweezers will be to trap and hold living specimens too large for laser-trapping (e.g., zebrafish embryos and cancer-derived spheroids) using a MFT. These trapped multi-cellular structures will be held free from mechanical contact for time-lapse microscopy, and will be distorted by the acoustic tweezers to test the effects of altered physical forces on embryo and organoid development. These experiments require trapping and tweezing forces large enough to change the shape of the embryo (far greater than the forces possible with optical tweezers). We will develop electrical controllability of the trapping location in 3D space so that the captured specimens may be: (1) moved from one location to another, (2) stretched or compressed for the characterization of the cell's elastic properties, (3) brought into contact with other cells or gene-containing liposomes. These will all be performed under electrical command without the need to move the ultrasonic tweezers mechanically. To optimize the development of ultrasonic tweezers as an enabling tool for biological experiments, two biological labs (led by the co-Investigators) will participate in the research from the start. They will receive successive versions of acoustic tweezers at the ends of the 6th, 18th, 30th, and 42nd month during the 4-year research period, and will use them to conduct the proposed experiments with transgenic embryos, cell spheroids and non-adherent circulating cells. The proposed biological experiments require manipulation of live cells in a liquid environment without any damage caused by the holding device. Such contact-free manipulation would be extremely difficult, if not impossible, without the proposed tweezers. The two biology labs will participate actively in a synergistic advancement of the tweezers, performing the biological experiments, and providing timely feedbacks, directing the PI's lab towards creating the most useful designs. Since MFT focuses acoustic energy on a very small spot and is capable of delivering acoustic energy through an intermediate solid, it can be incorporated into various microfluidic platforms for the management of cells, liquids, particles and proteins. The MFT's electrical controllability on the location and direction of the trapping force, combined with amenability of MFT being formed into an array, will allow the creation of complex biochemical assays and/or biomedical treatments at high throughput. The MFT's unprecedented capability of 3D capture and on-demand manipulation of microparticles/cells (of tens - hundreds of microns in diameter) will open up many new possibilities in cell study, gene transfection, juxtaposition and manipulation.